Gold-platinum / iron oxide nanozymes and their applications

Abstract

The present invention relates to the field of tissue culture technology, and more specifically to gold-platinum / iron oxide nanozymes and their applications. The present invention provides gold-platinum / iron oxide nanozymes, which are synthesized in one pot at room temperature, have simple reaction steps and a short reaction time, making them more suitable for large-scale production. Experimental results show that compared with other existing nanozymes, they have better detection effects and significantly lower detection limits, showing broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and in particular to gold-platinum / iron oxide nanozymes and applications thereof. Background Art

[0002] In the field of sensors and immunosensors, gold / platinum bimetallic nanomaterials are widely used due to the excellent catalytic ability of platinum and the optical properties of gold. However, the synthesis of gold / platinum bimetallic nanomaterials is time-consuming and complicated, especially in the large-scale synthesis stage, which is inconsistent with the industrial production process. Compared with pure noble metal nanomaterials, iron-based metal oxides form a core-shell structure with noble metal nanoparticles, which helps these noble metal nanoparticles maintain their original shape, size, surface state and catalytic activity. However, the synthesis of such iron-based noble metal nanoparticles usually requires harsh reaction conditions such as high temperature and high pressure, and has cumbersome steps, which greatly hinders their large-scale application.

[0003] Immunochromatographic test strips have the advantages of rapid, convenient, and low-cost detection, and have been widely used in food, environmental, and clinical marker detection. However, the commonly used gold nanoparticle detection sensitivity is low, and the detection limit is 2 to 3 orders of magnitude higher than that of the enzyme-linked immunosorbent assay with enzyme-linked immunosorbent enhancement. Therefore, drawing on the principles of enzyme-linked immunosorbent assay, applying the nanozyme signal enhancement strategy to immunochromatographic test strips can significantly improve the detection sensitivity. At present, there is still a gap in the technology of using nanozymes for signal enhancement on immunochromatographic test strips, and the existing nanozyme synthesis conditions are harsh and cumbersome. Therefore, it is crucial to develop new nanozymes that are more suitable for immunochromatographic systems and methods for preparing nanozymes, and the performance of existing immunochromatographic tests also needs to be further improved. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide gold-platinum / iron oxide nanozymes and their applications.

[0005] The present invention provides a gold-platinum / iron oxide nanozyme. The gold-platinum / iron oxide nanozyme has a nearly spherical morphology, an Au / Pt molar ratio of 3.35 to 32.35, an average particle size of 42 nm to 54 nm, and has catalase activity.

[0006] The present invention provides a method for preparing the gold-platinum / iron oxide nanozyme, which comprises the following steps:

[0007] A first reaction liquid is obtained by reacting a mixed solution of HAuCl4 and H2PtCl4 with aqueous ammonia;

[0008] The first reaction liquid undergoes a second reaction with FeCl2 to obtain gold platinum / iron oxide nanozyme.

[0009] Furthermore, in the preparation method,

[0010] In the mixed solution, the molar ratio of HAuCl4 and H2PtCl4 is (0.25-5):1;

[0011] The total amount of HAuCl4 and H2PtCl4 and the amount of ammonia water are in a ratio of 1:(4-8);

[0012] The total amount of HAuCl4 and H2PtCl4 and the amount of FeCl2 are in a ratio of 1:(5-10);

[0013] Further,

[0014] The total amount of HAuCl4 and H2PtCl4 and the amount of ammonia water are in a ratio of 1:6;

[0015] The total amount of HAuCl4 and H2PtCl4 and the amount of FeCl2 are in a ratio of 1:8.

[0016] In the present invention, the ratio of reagents is different, and the ion ratio structure of the synthesized nanozyme is different.

[0017] In some specific embodiments of the present invention, when the molar ratio of HAuCl4 and H2PtCl4 is 4:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water is 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 is 1:8, the synthesized sample is gold platinum / iron oxide nanozyme-4, and its Au / Pt value is 32.35;

[0018] In other specific embodiments of the present invention, when the molar ratio of HAuCl4 and H2PtCl4 is 1.5:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water is 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 is 1:8, the synthesized sample is gold platinum / iron oxide nanozyme-1.5, and its Au / Pt value is 13.05;

[0019] In other specific embodiments of the present invention, when the molar ratio of HAuCl4 and H2PtCl4 is 1:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water is 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 is 1:8, the synthesized sample is gold platinum / iron oxide nanozyme-1, and its Au / Pt value is 11.36;

[0020] In other specific embodiments of the present invention, when the molar ratio of HAuCl4 and H2PtCl4 is (2 / 3):1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water is 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 is 1:8, the synthesized sample is gold platinum / iron oxide nanozyme-2 / 3, and its Au / Pt value is 6.80;

[0021] In other specific embodiments of the present invention, when the molar ratio of HAuCl4 and H2PtCl4 is 0.25:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water is 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 is 1:8, the synthesized sample is gold platinum / iron oxide nanozyme-0.25, and its Au / Pt value is 3.35.

[0022] In the preparation method of the present invention, the conditions for the first reaction are mixing at 300 rpm to 600 rpm for 1 to 10 min, preferably mixing at 500 rpm for 2 min;

[0023] The second reaction is carried out at 300 rpm to 600 rpm for 10 to 60 min, preferably at 500 rpm for 20 min.

[0024] Furthermore, the second reaction further comprises a washing step, wherein the washing solution is deionized water. It should be noted that centrifugation between each independent washing is a conventional technical means used by those skilled in the art, and the present invention will not elaborate on this.

[0025] The gold-platinum / iron oxide nanozyme provided by the present invention is synthesized in one pot at room temperature, with simple reaction steps and short time. Compared with other nanozymes, it is more suitable for large-scale production. It is applied to immunochromatographic detection. Experimental results show that compared with other existing nanozymes, it has good detection effect and significantly reduced detection limit, and has broad application prospects.

[0026] The present invention provides a conjugate comprising at least one of the gold-platinum / iron oxide nanozyme of the present invention or the gold-platinum / iron oxide nanozyme prepared by the preparation method of the present invention and an immunochromatographic marker;

[0027] The detection marker includes at least one of an antigen, an antibody, an enzyme, DNA, RNA and / or a metabolite.

[0028] Furthermore, in a specific embodiment of the present invention, the immunochromatography marker is an antibody, specifically a cTnI monoclonal antibody, which is used as a representative to detect the effect of the nanozyme of the present invention in immunochromatography. The results show that the detection line of the nanozyme of the present invention for immunochromatography is lower, and it has good application prospects.

[0029] The present invention provides the use of at least one of the following I) to III) in the preparation of immunoassay products and / or biological assay products:

[0030] 1) the gold-platinum / iron oxide nanozyme of the present invention;

[0031] II), a mixture of gold-platinum / iron oxide nanozymes prepared by the preparation method of the present invention;

[0032] III), the conjugate of the present invention.

[0033] The present invention provides a test strip for immunoassay, which contains the conjugate of the present invention.

[0034] The present invention provides a kit comprising at least one of PBST, 3,3',5,5'-tetramethylbenzidine solution, sodium acetate / acetic acid buffer solution and the test strip of the present invention.

[0035] Furthermore, the kit of the present invention may also include other reagents, consumables, instruments or a combination thereof for immunochromatographic detection, which is not limited in the present invention.

[0036] The present invention provides an immunochromatographic detection method, which utilizes the test strip and / or the reagent of the present invention to detect a sample.

[0037] The present invention provides a gold-platinum / iron oxide nanozyme, which is synthesized in one pot at room temperature, has simple reaction steps and short time, and is more suitable for large-scale production; it is applied to immunochromatographic detection, and experimental results show that compared with other existing nanozymes, it has good detection effect and significantly reduced detection limit, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Transmission electron microscopy image of gold platinum / iron oxide nanozyme-1 synthesized by the method of Example 1;

[0039] Figure 2 1 shows the X-ray diffraction pattern of gold platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention;

[0040] Figure 3 The X-ray photoelectron spectrum of gold platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention is shown;

[0041] Figure 4The synthesis of gold platinum / iron oxide nanozyme-1 in Example 1 of the present invention is shown in Figure 1. The H2O2 concentration is kept constant (2 mol L -1 ), 3,3',5,5'-tetramethylbenzidine concentration from 0.05mmol L -1 to 0.6 mmol L -1 The Michaelis-Menten curve of the microplate reader measurement data;

[0042] Figure 5 The double reciprocal graph corresponding to the Michaelis-Menten equation for synthesizing gold platinum / iron oxide nanozyme-1 according to Example 1 of the present invention is shown;

[0043] Figure 6 The test results of the gold-platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention on the immunochromatographic test strip are shown, wherein A is a picture of the immunochromatographic test strip catalyzed by the nanozyme without gold-platinum / iron oxide nanozyme signal enhancement to detect PBS, myoglobin (Myo), creatine kinase isoenzyme (CK-MB), C-reactive protein (CRP), serum amyloid protein A (SAA), cTnI and a mixture of cTnI and other proteins; B is a picture of the immunochromatographic test strip after gold-platinum / iron oxide nanozyme signal enhancement. DETAILED DESCRIPTION

[0044] The present invention provides a preparation method and application of gold platinum / iron oxide nanozymes. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] This patent uses troponin as an example to demonstrate the excellent detection performance of gold-platinum / iron oxide nanozymes on immunochromatography. It is worth noting that this method is generally applicable to the detection of other markers by changing the linked antibody, which is just a feature of the immunochromatography platform.

[0046] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0047] The present invention will be further described below in conjunction with the embodiments:

[0048] Example 1 Preparation method of gold platinum / iron oxide nanozyme

[0049] The following steps are involved:

[0050] Step 1: Synthesize gold-platinum / iron oxide nanozymes using a redox self-assembly strategy;

[0051] Step 2: preparing a conjugate of gold platinum / iron oxide nanozyme and troponin (cTnI) antibody;

[0052] Step 3: Prepare immunochromatographic test strips for detecting cTnI;

[0053] Step 4: Immunochromatographic test strips detect cTnI through gold-platinum / iron oxide nanozyme signal enhancement.

[0054] According to the present invention, the step 1 is preferably specifically:

[0055] (1) First, H2PtCl4 solution, HAuCl4 solution and deionized water are mixed to obtain a mixed solution. The concentration of the HAuCl4 solution is preferably 0.01 to 0.05 mol L -1 , more preferably 0.01, 0.02, 0.03, 0.04, 0.05, or 0.01 to 0.05 mol L -1 The concentration of the H2PtCl4 solution is preferably 0.01 to 0.05 mol L -1 , more preferably 0.01, 0.02, 0.03, 0.04, 0.05, or 0.01 to 0.05 mol L -1 any value between; the molar ratio of the H2PtCl4 solution and the HAuCl4 solution is preferably 0.25:1 to 4:1.

[0056] (2) According to the present invention, an aqueous ammonia solution is rapidly added to the mixed solution under stirring at 500 rpm to obtain a reaction solution; the mass fraction of the aqueous ammonia solution is preferably 0.01% to 2%, more preferably 0.01%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, or any value between 0.01% and 2%. The ratio of the total amount of the HAuCl4 solution and the H2PtCl4 solution to the amount of the aqueous ammonia solution is preferably 1:6 to 1:15; the time for adding the aqueous ammonia solution and stirring is preferably 2 minutes, and the stirring speed is 500 rpm.

[0057] (3) Rapidly add FeCl2 solution to the reaction solution, stir the reaction, synthesize gold platinum / iron oxide nanozymes by self-oxidation reduction assembly method, centrifuge, wash with water 3 times, and disperse in water. The concentration of the FeCl2 solution is preferably 0.05-0.6 mol L -1 , more preferably 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.05 to 0.6 mol L -1any value between; preferably the total amount of HAuCl4 and H2PtCl4 and the amount of FeCl2 ratio is preferably 1:5 to 1:10; the FeCl2 solution is added and stirred for 20min.

[0058] (4) The reaction product solution in (3) was centrifuged and washed, and dispersed in deionized water to obtain a gold platinum / iron oxide nanozyme solution.

[0059] According to the present invention, step 2 is preferably:

[0060] 1 mL of gold platinum / iron oxide nanozyme solution was mixed with 100 μL of 1% sodium polyacrylate, centrifuged at 7500 rpm, and then dispersed in 1 mL of 10 mmol L -1 2-(N-morpholino)ethanesulfonic acid buffer solution, add 10 μL 100mmolL -1 1-ethyl-(3-dimethylpropyl)carbodiimide hydrochloride and 10 μL 100 mmol L -1 After 30 min of reaction, 10 μg of cTnI monoclonal antibody I was added. After 2 h of reaction, 10 μL of 10 mg mL -1 The Tris-HCl solution has a pH of 8.0 and contains 5% by mass of bovine serum albumin, 5% by mass of sucrose, and 0.5% by mass of Tween 20.

[0061] According to the present invention, the step three is preferably:

[0062] The cTnI monoclonal antibody II and goat anti-rabbit secondary antibody IgG were coated on the nitrocellulose membrane at a streak volume of 1 μL / cm as the detection line and quality control line, respectively, and then vacuum-dried at 37°C for 4 hours. The sample pad was pre-treated with a treatment solution (20 mmol L - 1 After being completely soaked in Tris-HCl (pH 8.0, containing 0.2% Tween 20, 2% bovine serum albumin, and 1% polyvinylpyrrolidone), the test strips were vacuum-dried at 37°C for 2 hours. The conjugate pad, sample pad, and absorbent pad were sequentially attached to the corresponding positions on the backing plate and cut into 4mm wide test strips. The conjugate of the gold-platinum / iron oxide nanozyme and the cTnI antibody was applied to the conjugate pad of the test strips to produce a cTnI detection test strip.

[0063] According to the present invention, the step 4 is preferably:

[0064] Add 100 μL of the sample solution to be tested to the sample pad, let it stand for 15 minutes until the test strip is fully reacted, and observe the color development of the test line and the quality control line. Continue to add 80 μL of PBST (PBS containing 0.05% Tween 20 by mass is PBST) to the sample pad to wash away the excess nanozymes on the test strip. Add 3,3',5,5'-tetramethylbenzidine substrate solution (the preparation method of the 3,3',5,5'-tetramethylbenzidine substrate solution: 6 μL of 3,3',5,5'-tetramethylbenzidine solution for membrane, 2 μL of 1.0 mol L -1 Sodium acetate / acetic acid buffer, mix well and adjust pH to 4.0, add 2 μL 10 mol L -1 The mixture was added dropwise to the test line and the quality control line, respectively, and allowed to stand at room temperature for 5 minutes. The color development of the test line and the quality control line was observed again.

[0065] The gold platinum / iron oxide nanozymes synthesized by the present invention are specifically shown in Table 1:

[0066] Table 1. Qualitative indicators and data of gold platinum / iron oxide nanozymes

[0067]

[0068]

[0069] When the sample gold platinum / iron oxide nanozyme-4 was synthesized, the molar ratio of HAuCl4 and H2PtCl4 was 4:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water was 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 was 1:8;

[0070] When the sample gold platinum / iron oxide nanozyme-1.5 was synthesized, the molar ratio of HAuCl4 and H2PtCl4 was 1.5:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water was 1:6; and the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 was 1:8.

[0071] When the sample gold platinum / iron oxide nanozyme-1 was synthesized, the molar ratio of HAuCl4 and H2PtCl4 was 1:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water was 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 was 1:8;

[0072] When the sample gold platinum / iron oxide nanozyme-2 / 3 was synthesized, the molar ratio of HAuCl4 and H2PtCl4 was (2 / 3):1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water was 1:6; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 was 1:8;

[0073] When the sample gold platinum / iron oxide nanozyme-0.25 was synthesized, the molar ratio of HAuCl4 and H2PtCl4 was 0.25:1; the molar ratio of the total amount of HAuCl4 and H2PtCl4 to ammonia water was 1:6; and the molar ratio of the total amount of HAuCl4 and H2PtCl4 to FeCl2 was 1:8.

[0074] 2. Comparison of Nanozyme Catalytic Activity

[0075] Taking the sample gold platinum / iron oxide nanozyme-1 as an example, the catalytic activity is compared with other nanozymes. The results are shown in Table 2:

[0076] Table 2. Comparison of the catalytic activity of the nanozyme of the present invention with other nanozymes

[0077] <![CDATA[K M (×10 -6 M)]]> <![CDATA[K CAT (×10 5 S -1 )]]> <![CDATA[K CAT / K M (×10 9 M -1 S -1 )]]> Horseradish catalase 434 0.04 0.0092 Iron oxide nanoparticles 98 0.32 0.326 platinum nanoparticles 102.980 3.024 2.937 Gold platinum / iron oxide nanozyme of the present invention 51.8 3.32 6.32

[0078] K M K is the Michaelis constant, which is the substrate concentration when the enzyme reaction is half the maximum reaction rate. The smaller the Km, the lower the substrate concentration required for the enzyme to react. It can also be said that the affinity between the enzyme and the substrate is greater. CAT is the conversion number, K CAT It measures the rate at which the enzyme catalyzes the production of substrate under optimal conditions. CAT / K M The ratio of the two constants is the most important parameter to measure the catalytic efficiency of an enzyme. CAT The larger the K M The smaller it is, the greater the ratio of the two is. CAT The larger the K, the faster the enzyme converts the substrate. M The smaller the value, the greater the affinity between the enzyme and the substrate.

[0079] The method of the present invention is simple and time-saving. The synthesis of nanozymes in patent CN 111007251 A involves high temperature and high pressure conditions, a reaction time of 14 hours, and harsh and cumbersome synthesis conditions. The nanozymes of the present invention differ significantly from existing nanozymes in elemental composition, synthesis method, and catalytic activity. The nanozymes of this patent are synthesized in one pot at room temperature. The synthesis method is simple and rapid, and the reaction time is less than 30 minutes, making them more suitable for large-scale production. The detection limit of the immunochromatographic system prepared in this patent is 0.05 ng / mL, which is about 2 orders of magnitude higher than that of CN 111007251A.

[0080] Example 2 Preparation and Application of Gold-Platinum / Iron Oxide Hybrid Nanozymes

[0081] Taking the sample gold platinum / iron oxide nanozyme-1 as an example,

[0082] Step 1 (Synthesis of gold-platinum / iron oxide nanozymes using redox self-assembly strategy):

[0083] (1) Add 2.5 mL of 0.024 mol L -1 HAuCl4 solution, 2.5 mL 0.024 mol L -1 Mix H2PtCl4 solution and 500mL deionized water evenly;

[0084] (2) quickly adding 20 mL of 0.075% ammonia aqueous solution to the mixed solution (1), and stirring at 500 rpm for 2 min to obtain a reaction solution;

[0085] (3) Add 10 mL of 0.1 mol L -1 FeCl2 solution was added to the reaction solution (2) and stirred at 500 rpm for 20 min;

[0086] (4) After the reaction is complete, the reaction solution obtained in (3) is centrifuged and washed three times at a centrifugal speed of 7500 rpm for 8 min, and dispersed in 100 mL of deionized water to obtain a gold platinum / iron oxide nanozyme solution.

[0087] Step 2 (preparation of a conjugate of gold platinum / iron oxide nanozyme and troponin (cTnI) antibody):

[0088] Take 1 mL of gold platinum / iron oxide nanozyme solution and mix it with 100 μL of 1% sodium polyacrylate, centrifuge at 7500 rpm and disperse it in 1 mL of 10 mmol L -1 10 μL 100 mmol L 2-(N-morpholino)ethanesulfonic acid buffer solution was added -1 1-ethyl-(3-dimethylpropyl)carbodiimide hydrochloride and 10 μL 100 mmol L -1After 30 min of reaction, 10 μg of cTnI monoclonal antibody I was added. After 2 h of reaction, 10 μL of 10 mg mL -1 The Tris-HCl solution has a pH of 8.0 and contains 5% by mass of bovine serum albumin, 5% by mass of sucrose, and 0.5% by mass of Tween 20.

[0089] Step 3 (Preparation of immunochromatographic test strips for cTnI detection):

[0090] The cTnI monoclonal antibody II and goat anti-rabbit secondary antibody IgG were coated on the nitrocellulose membrane at a streak volume of 1 μL / cm as the detection line and quality control line, respectively, and then vacuum-dried at 37°C for 4 hours. The sample pad was pre-treated with a treatment solution (20 mmol L - 1 After thorough immersion in Tris-HCl (pH 8.0, containing 0.2% Tween 20, 2% bovine serum albumin, and 1% polyvinyl pyrrolidone), the test strips were vacuum-dried at 37°C for 2 hours. The conjugate pad, sample pad, and absorbent pad were sequentially attached to the corresponding positions on the backing plate and cut into 4mm wide test strips. The gold-platinum / iron oxide nanozyme conjugate with the cTnI antibody was applied to the conjugate pad of the test strips to produce a cTnI detection test strip.

[0091] Step 4 (cTnI detection by immunochromatographic test strips using gold-platinum / iron oxide nanozyme signal enhancement):

[0092] Add 100 μL of the sample solution to the sample pad and let it stand for 15 minutes until the test strip is fully reacted. Observe the color development of the test line and the quality control line. Continue to add 80 μL of PBST (PBS containing 0.05% Tween 20 by mass) to the sample pad to wash away the excess nanozyme on the test strip. Add 3,3',5,5'-tetramethylbenzidine substrate solution (6 μL of 3,3',5,5'-tetramethylbenzidine solution for membrane, 2 μL of 1.0 mol L -1 Sodium acetate / acetic acid buffer, pH = 4.0, and 2 μL 10 molL -1 H2O2) was added dropwise to the test line and the quality control line respectively, and allowed to stand at room temperature for 5 minutes. The color development of the test line and the quality control line was observed again.

[0093] The transmission electron microscopy image of the gold platinum / iron oxide nanozyme-1 synthesized by the method of Example 1 is as follows: Figure 1 As shown, the results show that the morphology of the nanoparticles synthesized according to the method of Example 1 is spherical, and the average particle size is 48±5.3nm; the X-ray diffraction pattern of the gold platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention is as shown Figure 2 As shown, there are only diffraction peaks related to gold and platinum, but no diffraction peaks related to Fe, indicating that the formed iron oxide is amorphous. Figure 3 The X-ray photoelectron spectrum of the gold platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention is shown, indicating that the gold platinum / iron oxide nanozyme is composed of iron, oxygen, carbon, gold, and platinum elements. The performance and test results of the gold platinum / iron oxide nanozyme-1 when used for immunoassay are shown in FIG. Figure 4 、 Figure 5 and Figure 6 shown. Figure 4 The gold platinum / iron oxide nanozyme-1 of Example 1 of the present invention is kept at a constant H2O2 concentration (2 mol L -1 ), 3,3',5,5'-tetramethylbenzidine concentration from 0.05mmol L -1 to 0.6 mmol L -1 The Michaelis-Menten curve of the microplate reader measurement data; Figure 5 This is a double reciprocal graph corresponding to the Michaelis-Menten equation for synthesizing gold platinum / iron oxide nanozyme-1 in Example 1 of the present invention; Figure 6 This is a picture (Figure A) of the gold-platinum / iron oxide nanozyme-1 synthesized in Example 1 of the present invention detecting PBS, myoglobin (Myo), creatine kinase isoenzyme (CK-MB), C-reactive protein (CRP), serum amyloid protein A (SAA), cTnI and a mixture of cTnI and other proteins on an immunochromatographic test strip without nanozyme catalysis; and a picture (Figure B) of the immunochromatographic test strip after gold-platinum / iron oxide nanozyme signal enhancement.

[0094] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Gold platinum / iron oxide nanozyme, characterized in that The Au / Pt molar ratio is 6.80-11.36, the average particle size is 42 nm-54 nm, and the nanozyme has catalase activity. The preparation method of the gold-platinum / iron oxide nanozyme comprises the following steps: A first reaction liquid is obtained by reacting a mixed solution of HAuCl4 and H2PtCl4 with aqueous ammonia; The first reaction solution is subjected to a second reaction with FeCl2 to obtain gold platinum / iron oxide nanozyme; In the mixed solution, the molar ratio of HAuCl4 and H2PtCl4 is (2 / 3~1):1; The total amount of HAuCl4 and H2PtCl4 and the amount of ammonia water are in a ratio of 1:(4-8); The total amount of HAuCl4 and H2PtCl4 and the amount of FeCl2 are in a ratio of 1:(5-10).

2. The method for preparing the gold platinum / iron oxide nanozyme according to claim 1, characterized in that: The steps include: A first reaction liquid is obtained by reacting a mixed solution of HAuCl4 and H2PtCl4 with aqueous ammonia; The first reaction solution is subjected to a second reaction with FeCl2 to obtain gold platinum / iron oxide nanozyme; In the mixed solution, the molar ratio of HAuCl4 and H2PtCl4 is (2 / 3~1):1; The total amount of HAuCl4 and H2PtCl4 and the amount of ammonia water are in a ratio of 1:(4-8); The total amount of HAuCl4 and H2PtCl4 and the amount of FeCl2 are in a ratio of 1:(5-10).

3. The preparation method according to claim 2, characterized in that The first reaction was performed at 300-600 rpm for 1-10 min; The second reaction was performed at 300-600 rpm for 10-60 min.

4. The preparation method according to claim 2 or 3, characterized in that The conditions for the first reaction were 500 rpm, 2 min; The conditions for the second reaction were 500 rpm, 20 min.

5. The preparation method according to claim 4, characterized in that The second reaction is followed by a washing step, wherein the washing solution is deionized water.

6. A conjugate, characterized in that Comprising at least one of the gold-platinum / iron oxide nanozyme according to claim 1 or the gold-platinum / iron oxide nanozyme prepared by the preparation method according to any one of claims 2 to 5 and an immunochromatographic marker; The immunochromatographic marker includes at least one of an antigen, an antibody, an enzyme, a DNA, an RNA and / or a metabolite.

7. The conjugate according to claim 6, characterized in that The immunochromatographic marker is an antibody.

8. Use of at least one of the following I) to III) in the preparation of immunoassay products and / or biological assay products: 1), the gold platinum / iron oxide nanozyme according to claim 1; II), gold platinum / iron oxide nanozyme prepared by the preparation method according to any one of claims 2 to 5; III), the conjugate according to claim 6 or 7.

9. A test strip for immunoassay, characterized in that: Containing the conjugate according to claim 6 or 7.

10. A kit, characterized in that The invention comprises at least one of PBST, 3,3',5,5'-tetramethylbenzidine solution, sodium acetate / acetic acid buffer solution and the test strip according to claim 9.

Images